Clonal analysis of the BALB/c T cell proliferative response to apo beef cytochrome c.

Murine T cell clones that proliferated specifically in response to the protein antigen apo cytochrome c were derived and maintained in continuous culture. Two distinct clonotypes were observed with respect to the proliferative responses observed when a variety of peptides prepared from several species of cytochrome c were tested. These 2 clonotypes appeared to recognize 2 different regions in the cytochrome c molecule. Only 1 of the 2 clonotypes tested demonstrated helper cell activity for antibody formation in vitro

Transduction of CpG DNA-stimulated primary human B cells with bicistronic lentivectors.

Recently, using HIV-1-derived lentivectors, we obtained efficient transduction of primary human B lymphocytes cocultured with murine EL-4 B5 thymoma cells, but not of isolated B cells activated by CD40 ligation. Coculture with a cell line is problematic for gene therapy applications or study of gene functions. We have now found that transduction of B cells in a system using CpG DNA was comparable to that in the EL-4 B5 system. A monocistronic vector with a CMV promoter gave 32 +/- 4.7% green fluorescent protein (GFP)+ cells. A bicistronic vector, encoding IL-4 and GFP in the first and second cistrons, respectively, gave 14.2 +/- 2.1% GFP+ cells and IL-4 secretion of 1.3 +/- 0.2 ng/10(5) B cells/24 h. This was similar to results obtained in CD34+ cells using the elongation factor-1alpha promoter. Activated memory and naive B cells were transducible. After transduction with a bicistronic vector encoding a viral FLIP molecule, vFLIP was detectable by FACS or Western blot in GFP+, but not in GFP-, B cells, and 57% of sorted GFP+ B cells were protected against Fas ligand-induced cell death. This system should be useful for gene function research in primary B cells and development of gene therapies

Anti-granulocyte opsonic activity in sera from patients with systemic lupus erythematosus.

Neutropenia is common in patients with systemic lupus erythematosus (SLE) but mechanisms of cell depletion remain obscure. To investigate the possible autoimmune aetiology of neutropenia in SLE, sera from 31 patients with this disorder were tested for anti-granulocyte activity. Granulocyte-binding immunoglobulins were detected by indirect immunofluorescence, and the ability of patient sera to opsonize granulocytes was determined by measuring the chemiluminescent response of human monocytes to granulocytes sensitized by test sera. Sera from 22 of the 31 patients bound IgG to granulocyte cell membranes and/or to nuclei, but only membrane-binding antibodies opsonized the cells for recognition by monocytes. There was no correlation between neutrophil count and the level of granulocyte-binding IgG as measured by indirect immunofluorescence. In contrast, opsonic activity and neutrophil count were inversely correlated (r = 0.5; P less than 0.05). However, opsonic activity was present in sera from most non-neutropenic patients. In patients with SLE, impaired reticuloendothelial system function may allow sensitized granulocytes to remain in the circulation